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Seamless insert-plasmid assembly at high efficiency and low cost

Benoit, Roger, Ostermeier, Christian, Geiser, Martin, Widmer, Hans, Auer, Manfred and Li, Julia Su Zhou (2016) Seamless insert-plasmid assembly at high efficiency and low cost. PLOS ONE, April . pp. 1-13.


Seamless cloning methods, such as sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the construction of protein expression plasmids. We here show that single-stranded gaps in double-stranded plasmids, which for example occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Conversely, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These data point out a critical factor for robust seamless cloning. Highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this method with two short insert-plasmid overlap regions, each counting only 15 nucleotides.

Item Type: Article
Date Deposited: 03 May 2016 23:45
Last Modified: 03 May 2016 23:45


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